USING QPCR TO UNDERSTAND UP AND DOWN REGULATION OF ANTIBIOTIC RESISTANCE GENES IN SALMONELLA HEIDELBERG CHALLENGED WITH COMMONLY USED ANTIMICROBIALS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1015826
• Project Start Date: Apr 4, 2018
• Project End Date: Sep 30, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Population Health & Reproduction

Non Technical Summary
Salmonella is a significant food-borne pathogen that causes an estimated one million foodborne illnesses, 19,000 hospitalizations and 380 deaths per year in the United States.In general, antibiotics are not used to treat non host-adapted (i.e. carried sub-clinically in poultry and cause disease in humans) Salmonella outbreaks in poultry. However, antimicrobials like ionophores, bacitracin (BMD), copper and zinc are commonly used prophylactically in the conventional poultry industry to prevent or mitigate various other potential infectious diseases including coccidiosis, E. coli and Clostridium perfringens. While antibiotic resistance has become a major threat to food safety and public health, our understanding of how bacteria become antibiotic resistant is primarily focused on mutation, selective pressure from antibiotic usage and plasmid based horizontal gene transfer of resistance genes.However, other mechanisms of resistance may play a significant role. Specifically,non-antibiotics have the potential to affect microbial resistance by merely up-regulating expression of genes activated by exposure to certain disinfectants. Results of this project will include a detailed assessment of anibiotic resistance patterns and resistant gene expression from antibiotics and other agents (Cu, Zn, disinfectants) that cause antimicrobial resistance.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3299	1100	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

food safety

antimicrobial resistance

poultry

Goals / Objectives
The goal of this proposed project is to use genotypic (i.e. qPCR) and phenotypic (i.e. disc diffusion) assays to gain a better understanding of the effect they may have on antibiotic resistance genes. Specific objectives are: 1.) To optimize a RT q-PCR assays for the 6 resistance genes selected from our previous RNA-seq work 2.) To determine statistically significant up and down regulation of the 6 resistance genes using qPCR; 3.) To characterize antibiotic resistance of Salmonella Heidelberg (SH)exposed to these antimicrobials using the disc diffusion assay.

Project Methods
Two Salmonella Heidelberg (SH)field were originally isolated from a commercial processing plant in 2014 and 1992 respectively. From our previous RNA-seq work 98% of the genes aligned to a publically available SH reference genome. In addition from our previous RNA-seq work we identified 6 genes that had functions associated with antimicrobial resistance. Using those genes we will develop primers and probes for individual qPCR assays. Next we will grow cultures of the SH strains to mid-log phase and expose them individually to realistic doses of the ionophore Salinomycin, BMD, copper and zinc. RNA will be extracted using a commercial kit and then 6 individual qPCR assays will be performed for each resistance gene. Using the Delta-Delta Cq method the fold increase or decrease in gene expression will be measured comparing the exposed SH to the control SH. Assays will be done in triplicate.Antibiotic resistance of SH will be determined by bacterial susceptibility of the exposed SH to antimicrobial drugs commonly used in broiler farms. Thirty individual colonies of each SH strain will be challenged with serially diluted antibiotics that are medically important in humans including Aminoglycosides, Penicillins, Tetracyclines Cephalosporins, and Quinolones, using the minimum inhibitory concentration (MIC) method. Resistance (R), intermediate resistance (IR), and susceptible (S) will be interpreted by the standards of susceptibility tests of enteric bacteria from animals (M31-A3) established by the ClinicalThe delta-delta or Livak method will be used to determine the fold-up or down regulation between the control samples and treated samples. Relative quantification using Cq is a method that is extensively used.

Progress 04/04/18 to 09/30/18

Outputs
Target Audience:Academics and food safety professionals (with a focus on the poultry industry) who are keen to understand different aspect of antimicrobial resistance. Changes/Problems:Change: It was less expensive and scientifically more sound to switch from qPCR to RNA-seq of the entire genome. The advantage of this technique (e.g. RNA seq) is that you get whole genome results as opposed to results from selected genes using qPCR. What opportunities for training and professional development has the project provided?A graduate student performed the majority of the laboratory work and worked with the UC Davis Genome Center to do the associated bioinformatics work. How have the results been disseminated to communities of interest?yes the results were shared at the Western Poultry Disease (WPDC) and the American Association of Avian Pathologists (AAAP)annual meetings What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This research showed that the transcriptome (RNA) of the same species of Salmonella Heidelberg (SH) respondes differently to 3 different disinfectants. The importance of this is that for example exposure of SH to acidified Calcium Hypochlorite (aCH) will cause an upregulation of genes related to antimicrobial resistance and virulence which not be static or cidal to SH at the levels of disinfectant used.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: MYRNA CADENA, LUTZ FROENICKE, MONICA BRITTON, MATTHEW L. SETTLES, BLYTHE DURBIN-JOHNSON, EMILY KUMIMOTO, RODRIGO A. GALLARDO, AURA FERREIRO, TEREZA CHYLKOVA, HUAIJUN ZHOU, and MAURICE PITESKY (2019) Transcriptome Analysis of Salmonella Heidelberg after Exposure to Cetylpyridinium Chloride, Acidified Calcium Hypochlorite, and Peroxyacetic Acid. Journal of Food Protection: January 2019, Vol. 82, No. 1, pp. 109-119. https://doi.org/10.4315/0362-028X.JFP-18-235

Progress 04/04/18 to 09/30/18

Outputs
Target Audience:Commercial poultry industry and public health officials concerned about food safety and the use of disinfectants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This work was the major thesis work of a MS graduate student in Avian Sciences. She was able to learn basic micorbiology, molecular biology and bio-informatics. How have the results been disseminated to communities of interest?At various extension based and scientificmeetings What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The resulting reserach identified what commonly used disinfectants are effective at reducing expression of virulence and pathogenicity genes in Salmonella Heidelberg. The results and methodology could lead to new insights in food safety control where instead of just considering kill curves, producers would also consider living bacteria and how those bacteria are effected by disinfectants,

Publications

• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Cadena, Myrna, Lutz Froenicke, Monica Britton, Matthew L. Settles, Blythe Durbin-Johnson, Emily Kumimoto, Rodrigo A. Gallardo et al. "Transcriptome Analysis of Salmonella Heidelberg after Exposure to Cetylpyridinium Chloride, Acidified Calcium Hypochlorite, and Peroxyacetic Acid." Journal of food protection 82, no. 1 (2018): 109-119.